Project Summary/Abstract Sjgren?s syndrome (SS) is a systemic autoimmune disease that predominantly affects women, yet the mechanisms driving this strong gender bias are unknown. The X chromosome has been implicated in the increased female susceptibility to autoimmune diseases like SS, and compelling evidence suggests that abnormal X chromosome inactivation (XCI) may play a pathogenic role. XCI occurs during development to normalize X-linked gene dosage between men and women in a manner dependent on the X-inactive specific transcript (XIST), a long non-coding RNA (lncRNA). Although this is the only known function of XIST RNA, it continues to be synthesized throughout life in individuals with two X chromosomes. A non-classical distribution of XIST outside of the nucleus has recently been observed in a subset of B and T cells from healthy women. The mechanisms and consequences of this non-canonical XIST expression have not been explored, and several additional intriguing observations suggest that XIST possesses properties that may contribute to the pathogenesis of autoimmune diseases, independently of XCI, including: 1) XIST is a lncRNA present at high levels in individuals predisposed to the development of autoimmunity; 2) the dominantly targeted autoantigens in SS, Ro/SSA and La/SSB, are RNA-binding proteins that can be found in complexes with self-RNA; and 3) our analysis of the XIST RNA sequence reveals the presence of a known TLR7-stimulatory motif. Together with growing evidence that chronic exposure to self-RNA alone or in immune complexes can stimulate pathogenic TLR7-dependent responses, these observations support our hypothesis that XIST RNA contributes to the female susceptibility to autoimmunity by acting as an endogenous TLR7 agonist. We propose to build on our preliminary data to systemically examine the critical aspects of this hypothesis through the study of human biospecimens and cellular models. In Aim 1, high- throughput flow cytometric methods will be used to compare the expression and distribution pattern of XIST RNA in the peripheral blood cells of men and women with SS compared to sex-matched healthy controls. Aim 2 will examine XIST release from dying cells and potential interactions with SS autoantigens, to determine whether XIST could be a component of circulating immune complexes in patients with SS. Finally, in Aim 3, we will evaluate the capacity of XIST RNA to act as a TLR7 ligand. This work has the potential to define a novel proinflammatory role for XIST in the pathogenesis of SS, which could inform the future development of therapies that disrupt the disease promoting capacity of XIST for the treatment of SS and related autoimmune conditions.